For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Supplied in 10mM sodium HEPES (pH 7.5), 150mM NaCl, 100 μg/ml, 50% glycerol and less than 0.02% sodium azide. Store at -20ºC. Do not aliquot the antibody.
C-Reactive Protein (D1N1U) Rabbit mAb recognizes endogenous levels of total human C-reactive protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val38 of human C-reactive protein.
C-reactive protein (CRP) is a pentraxin family protein involved in several host defense-related functions as a result of its ability to bind to foreign pathogens and damaged host cells (1). CRP is a cyclic, non-covalent pentameric protein and normal constituent of human sera that is produced primarily by hepatocytes (2). Secretion of CRP is induced by proinflammatory cytokines, including IL-6 and IL-1β, and significantly increases during acute phase responses to tissue injury, infection, or other inflammatory stimuli (3,4). The presence of CRP is often utilized as an inflammation marker, and monitoring CRP levels in plasma is a useful tool in assessing disease progression or treatment effectiveness. CRP is also regarded as a risk assessment factor for the development and progression of cardiovascular disease (5).
CRP binds to phosphorylcholine that is present on the surface of damaged tissues and in the bacterial cell wall of certain pathogens (6). Through this calcium-dependent interaction, CRP promotes agglutination and initiates the activation of the complement cascade. This results in enhanced opsonization through CRP interaction with FcγRI and FcγRIIA, which facilitates phagocytosis (7).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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